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Molecules 2008, 13, 2190-2219; DOI: 10.

3390/molecules13092190
OPEN ACCESS

molecules
ISSN 1420-3049 www.mdpi.org/molecules Review

Polyphenols in Cocoa and Cocoa Products: Is There a Link between Antioxidant Properties and Health?
Abbe Maleyki Mhd Jalil and Amin Ismail * Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia 43400, UPM Serdang, Selangor Darul Ehsan, Malaysia * Author to whom correspondence should be addressed; E-mail: amin@medic.upm.edu.my; Tel.: +603- 89472435; Fax: +603-89426769. Received: 16 August 2006; in revised form: 29 August 2008 / Accepted: 2 September 2008 / Published: 16 September 2008

Abstract: Cocoa and cocoa products have received much attention due to their significant polyphenol contents. Cocoa and cocoa products, namely cocoa liquor, cocoa powder and chocolates (milk and dark chocolates) may present varied polyphenol contents and possess different levels of antioxidant potentials. For the past ten years, at least 28 human studies have been conducted utilizing one of these cocoa products. However, questions arise on which of these products would deliver the best polyphenol contents and antioxidant effects. Moreover, the presence of methylxanthines, peptides, and minerals could synergistically enhance or reduce antioxidant properties of cocoa and cocoa products. To a greater extent, cocoa beans from different countries of origins and the methods of preparation (primary and secondary) could also partially influence the antioxidant polyphenols of cocoa products. Hence, comprehensive studies on the aforementioned factors could provide the understanding of health-promoting activities of cocoa or cocoa products components. Keywords: Cocoa; polyphenols; antioxidants; methylxanthines

Introduction The chronology of cocoa began in 2,000 B.C, the date attributed by historians to the oldest drinking cups and plates that have ever been discovered in Latin America at a small village in the Ulúa valley in Honduras, where cocoa played a central role. In 200-900 AD, cocoa was one of the main products in

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Mayan agriculture and religion. For instance, cocoa is used as a gift to deceased dignitaries at their funeral ceremonies and as currency [1]. The word cacao is derived from the Olmec and the subsequent Mayan languages (kakaw) and the chocolate-related term cacahuatl is Nahuatl (Aztec language) derived from Olmec/Mayan etymology [2]. In 1737, the cocoa tree was named Theobroma cacao which refers to the mythical background of the tree literally means “cocoa, food of the gods” [1]. Dillinger et al. [2] reported that medicinal uses of cocoa had been traced from Mexican (Aztec) sources and approximately 150 uses of cocoa for medical treatment had been documented. Various parts of Theobroma cacao have been utilized, namely cocoa beans prepared as chocolate, cocoa bark, cocoa butter, cocoa flower, cocoa pulp and cocoa leaf. Cocoa was brought to Europe by the Spanish in 1505. By 1653, cocoa was used in Europe as a medicine rather than as a delicious foodstuff. The use of chocolate was recognized as stimulating the healthy function of the spleen and other digestive functions. Moreover, in the 17th and 18th century, chocolate was regularly prescribed or mixed into medications for all sorts of ailments and diseases from colds and coughing, to promote digestion, fertility, reinforce mental performance and as an anti-depressant [1]. Studies on the health benefits of cocoa and cocoa products have been conducted over the past decade, with a major focus on degenerative diseases. These benefits could be due to their significant amounts of flavonoid monomers (catechin and epicatechin) up to tetradecamers [3, 4]. It was noted that all polyphenols possessed antioxidant action in vitro, but do not necessarily exert antioxidant potential in vivo [5]. Most of the early studies focusing on health benefits of cocoa polyphenols came from human clinical trials [5]. Moreover, the study on health benefits of cocoa was not limited to that of human intervention but had also been extensively studied in vitro and in vivo [6-10]. The majority of the studies have examined the contributions of the flavonoids in cocoa and cocoaproducts towards health benefits, but it must be noted that cocoa and their products are also rich in methylxanthines, namely caffeine, theobromine and theophylline [11, 12] and studies have demonstrated that methylxanthines can possess both positive and negative health effects. For instance, caffeine intake has been reported to have negative effects on reproductive health [13]. On the other hand, caffeine supplementation enhanced net hepatic glucose uptake through increment of glucose-6phosphate production in the liver [14]. Cocoa is also rich in micronutrients [15] and micronutrients such as copper found in cocoa could contribute significantly towards human dietary intake [16]. As cocoa contains a mixture of bioactive components, it is possible to postulate that there may be direct or indirect synergism between these components in delivering their health properties. Cooper et al. [5] suggested that if the biological effects are due to cocoa flavonoids rather than the other components, the perfect control would be cocoa or cocoa products that contain things other than flavonoids. Factors affecting quality and quantity of cocoa and cocoa-based products during production and manufacturing are of great importance in delivering the best health effects. These factors could significantly reduce the polyphenol content of the selected products. Physiological factors, namely bioavailability and antioxidant properties, should also be considered when assessing their contribution in the biological system. For instance, there could be a significant correlation between interactions of polyphenols and proteins, but the interference of protein with polyphenols remains inconclusive, as some studies have demonstrated that protein could reduce the activity of polyphenols and some studies not [17, 18]. To date, there is a lack of studies which concurrently determine the outcomes and the bioavailability or antioxidant status of the studied subjects. Studies reported that antioxidant status of

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the subjects are not improved or enhanced although there were positive health outcomes. Hence, is there actually a link between cocoa antioxidants and health or is it due to any other components of the cocoa and cocoa products? Factors such as bioavailability, types of cocoa and cocoa products used in the intervention study, antioxidant status, and the state of subjects being studied (normal, borderline, or with disease) would at least affect the measured outcomes. Polyphenols and other components in cocoa beans and cocoa-based products Polyphenols Cocoa had long been identified as a polyphenols-rich food. The main polyphenol in cocoa or known as cacao was first identified by Ultée and van Dorsen in 1909 [19]. The crystalline compound they discovered, with empirical formula C16H16O6, was called “Kakaool”. For over 20 years afterwards, there was disagreement between researchers in naming this phenolic compound. Further purification indicated that this compound was catechin, with empirical formula C15H14O6 [20]. However, this compound was mistakenly named l-acacatechin as one of catechins present in the cutch-producing acacias (Acacia catechu). A year later, Freudenberg et al. [21] reported that “Kakaool” probably represented l-epicatechin, which could also be found in Acacia catechu. Later, they agreed that the name l-acacatechin was incorrect as both catechin and epicatechin are stereoisomers. Forsyth [22] reported that cocoa bean contains four types of catechins, of which (-)-epicatechin constitutes about 92%. Adam et al. [23] indicated that unfermented cocoa bean contains both tannin and catechin. The brown and purple color of the cocoa bean was attributed to the complex alteration products of catechin and tannin. Beside these compounds, cocoa was found to have leucoanthocyanins that are present as glycosides. Similarly, it was observed that cocoa beans contains two cyanidin glycosides and at least three leucocyanidins (procyanidin) compounds [22]. Leucocyanidins constitute about 60% of total polyphenols in fresh cocoa cotyledon [24]. Quesnel [25] found that cocoa bean contains simple dimeric leucocyanidin and epicatechin. Epicatechin and simple leucocyanidins 1, 2 and 3 (L1, L2, and L3) are present in cocoa beans, as identified using two-dimensional paper chromatography. In addition, cyanidin is also present as cyanidin-arabinoside and cyanidin-galactoside. Later in 1977, Jalal et al. [26] indicated that the major components of cocoa extracts (leaves, cotyledons, stem, and callus) are anthocyanins, leucocyanidins, (-)-epicatechin, catechin, p-coumaryl quinic acid and chlorogenic acid. The study on cocoa polyphenols became more extensive with the discovery of major low molecular weight polyphenols in cocoa, namely catechin, epicatechin, dimers epicatechin-(4β→8)-catechin (procyanidin B1), epicatechin-(4β→8)-epicatechin (procyanidin B2), and trimer [epicatechin(4β→8)]2-epicatechin (procyanidin C1) [27]. Previous studies showed that monomeric polyphenols, namely epicatechin and catechin, dimer, trimer, and tetramer were detected by reverse-phase liquid chromatography mass spectometry (RP LC-MS) [28]. It has been reported that flavonols (epicatechin and catechin) were predominant compounds in cocoa powder [29, 30]. Epicatechin was predominant in all chocolates, with a ratio of 1:0.1, compared to catechin [31]. The basic structure of flavanols is shown in Figure 1. Structures of the monomeric catechin and epicatechin enantiomers are shown in

Structures the catechin and epicatechin enantiomers [34. (+)-Catechin (2R. Figure 2. 3S) . Basic structure of flavonols [34]. 3R) (+)-Epicatechin (2S. Moreover. The chemical structures of flavonols and procyanidins are important for their antioxidant activity as they possess both free radical trapping and chelation of redox-active metals properties [35]. the interflavan bond at position 4 is always trans to the hydroxyl (OH) group at position 3. 3S) (-)-Epicatechin (2R. Flavonoids and procyanidins were found to prevent lipid oxidation through interaction between lipid forming membranes and the adsorption to the polar lipid headgroups [36]. 3R) (-)-Catechin (2S. Figure 1.Molecules 2008. The monomers are stereoisomers at position 3 of the C-ring. 46]. their respective enantiomers namely (–)-catechin and (+)-epicatechin are not commonly found in nature [34]. while the dimer and trimer are given in Figure 3. 13 2193 Figure 2. However. * indicates chiral center located at the C2 and C3-position of the C-ring. (+)-Catechin and (–)-epicatechin forms are commonly found in cocoa [33]. but have the same configuration at position 2 [32].

However. Over the past decade. deoxyclovamide. Recently.89-43. clovamide. epicatechin-(4β-8)-epicatechin Trimer C1. it is important to .Molecules 2008. As reported. 2194 Dimer B2.4α →8)-epicatechin. Structures of procyanidin dimer and trimer in cocoa [36]. 3T-O-β-D-galactopyranosyl-ent-epicatechin-(2α→7.4β→6)-epicatechin. These findings were attributed to antioxidant flavonoids ranging from monomers to oligomers in cocoa and chocolates as discussed before. which comprises 12-18% of the whole beans dry weight [38]. bioavailability of ingested flavonoids present in cocoa or cocoa-based products is of great importance as it may in turn reflect antioxidant status of the studied subjects. 43]. Cocoa was reported to have high polyphenols content. 45]. Thus. epicatechin-(4β→8)]2-epicatechin Porter et al. such as (–)-epicatechin (EC). (–)Epicatechin content in freshly prepared beans ranged from 21. with 610 mg total catechins/kg of fresh edible weight [43]. [37] confirmed that another three new compounds are present in fresh cocoa beans. Kelm et al. Kim and Keeney [44] developed a sensitive method for determination of (-)epicatechin in cocoa beans which was detected at 280 nm and quantified by using external standards. high performance liquid chromatography (HPLC) had been utilized in the determination of polyphenol compounds in cocoa. A study reported that in raw cocoa beans. it has been showed that chocolate is one of the most polyphenol-rich foods along with tea and wine [42. and oligomers of these monomeric base units. most of the outcomes were based on the short-term effects (between 4 days to 6 weeks) [5].4α→8)-epicatechin and 3T-O-L-arabinopyranosyl-ent-epicatechin(2α→7. at least 28 studies have been reported on the health benefits of cocoa flavonoids [5]. With advancements in technology. (+)-catechin.27 mg/g dry defatted samples [40]. 13 Figure 3. Cocoa is rich in polyphenols such as (+)-catechin. and anthocyanidins [41]. Chocolates is also rich in polyphenol substances. quercetin (including its glucoside). Is there a link between the antioxidant flavonoids of cocoa and cocoa products on health effects based on short-term studies? Hence. [3] later indicated that unfermented cocoa beans contain monomers up to tetradecamers. The new compounds were identified as epicatechin-(2β→5. trans-resveratrol and its glucoside (trans-piceid) and procyanidin [41. namely procyanidins. long-term feeding trials of cocoa and cocoa products on health benefits are warranted. 60% of total phenolics were flavonol monomers (epicatechin and catechin) and procyanidin oligomers [39]. (–)-epicatechin. Most studies showed positive relationships between cocoa and chocolate flavonoids on cardioprotective effects. Results indicated that dark chocolate exhibited the highest polyphenol content.

however. Cocoa supplementation exerts promising health properties due to its antioxidative properties. However. there were no changes in total antioxidant capacity and oxidative stress biomarker (8isoprostane). Moreover. Similarly.Molecules 2008. blood pressure [58]. All polyphenols possess antioxidant properties in vitro but are not likely to exert the same properties in vivo and in human. Similarly. Similarly. arterial flow mediated dilation (FMD) [59] and platelet aggregation [52]. The results indicated that a single dose of cocoa drink increased nitric oxide in human plasma and improved endothelial dysfunction. epicatechin exhibited dose-dependence in plasma after dark chocolate consumption as low as 1 nM [50]. heart rate and glycaemic control among diabetics. Most of the studied outcomes of cocoa polyphenols on cardiovascular health were on the antioxidant status. [63] reported that cocoa ingestion improved basal FMD by 30% without changes in endothelial function. Numerous studies have been reported on the health benefits of cocoa and their products on cardiovascular diseases [5]. cocoa intake decreased LDL oxidation without changes in antioxidant potentials and oxidative stress level in plasma [53]. low density lipoprotein (LDL) oxidation [57]. The measured outcomes were mainly focused on plasma antioxidant activity [51]. Flow mediated dilation (FMD) of the brachial artery is a tool for measuring endothelium-dependent dilation [62]. These studies clearly indicated that cocoa administration did not exert their antioxidative properties in plasma of healthy subjects. Wang et al. Adamson et al. These factors were associated with coronary diseases [61]. Supplementation of cocoa for four weeks significantly improves platelet function among healthy subjects [52]. [51] demonstrated that dark chocolate dose-dependently increased plasma antioxidants and decrease 8-isoprostane. Effects of monomers up to decamers derived from cocoa was dosedependent and prevented erytrocyte hemolysis in vitro and enhanced plasma antioxidant capacity [47]. there was no correlation between cocoa intake and plasma antioxidant status. although there were significant health outcomes. However. Milk chocolate bar consumption increased HDL levels compared to highcarbohydrate snacks among young men [56]. Most of the studies showed cocoa enhanced flow mediated dilation. blood pressure. Wan et al. nitric oxide bioactivity and platelet function [60]. inflammatory production. [5] reported that healthy subjects may already have optimum dietary status and supplementation will not produce meaningful outcomes. Dark chocolate supplementation for three weeks in healthy subjects significantly increased highdensity lipoprotein cholesterol (HDL-c) compared to their unsupplemented counterparts [54]. [55] demonstrated that cocoa powder and dark chocolate supplementation improved HDL levels by 4% compared to control diet. Physiologically. This could be due to the status of subjects recruited in the study. endothelial function. Balzer et al. but there were no changes in oxidative stress biomarkers. Cooper et al. The presence of epicatechin (12-fold from baseline) leads to significant increase in plasma total antioxidant capacity and decrease in plasma thiobarbituric acid reactive substances. The measurement of plasma antioxidant concentration and oxidative stress levels are examples of determining antioxidant status. there are also studies which failed to show these effects. An in vivo study indicated that epicatechin from cocoa could enhance the antioxidative activity of plasma [49]. Procyanidins extracted from cocoa exhibited endothelium-dependent . [48] indicated that polyphenol content positively correlated with antioxidant properties as measured by oxygen radical absorbance capacity (ORAC). 13 2195 consider both bioavailability and antioxidant status in determining the relationship between cocoa flavonoids and health benefits.

[53] demonstrated reduced LDL oxidation after cocoa supplementation for 6 weeks compared to unsupplemented subjects. 13 2196 relaxation (EDR) through activation of nitric oxide synthase activity in rabbit aortic rings in vitro [64]. Long-term supplementation of cocoa powder (12-weeks) to normo and mildly hypercholesterolemic human subjects had decreased LDL oxidation and increased plasma HDL cholesterol compared to the control group [68]. Amin et al. The results were reported to be due to the tetramers and higher polymers of epicatechin. Unlike cardiovascular diseases. and their CAD burden. and trimers were not capable of contributing to EDR. 72]. cocoa powder and cocoa liquor suppressed the development of atherosclerotic lesions and inhibited atherosclerosis [9. 68]. there was a study which showed negative association between flavonoids-rich chocolate consumption and cardiovascular diseases among coronary artery disease (CAD) subjects [66]. no changes were observed in the glycaemic index. [75] reported that incorporation of cocoa powder as flavour in six different foods (chocolate bars. [76] indicated that cocoa liquor extract lower the activity of tumor marker enzymes during hepatocarcinogenesis. there were no significant changes in LDL oxidation. systemic arterial compliance and forearm blood flow. Baba et al. 8-isoprostane. ice creams. High-flavonoid content dark chocolate (containing 259 mg polyphenols) significantly improved FMD in healthy subjects compared to low-flavonoid chocolate (containing trace amount of polyphenols) [65]. Although the supplementation of high-flavonoids dark chocolate increased plasma epicatechin. dimers. FMD. In human subjects with hypertension. 10]. More work need to be done in exploring the effects of cocoa polyphenols on diabetes risk. In contrast. there is still limited human study on the effects of cocoa polyphenols and diabetes in human clinical trials. In vivo studies indicated that dark chocolate. breakfast cereals. There were no significant changes observed in lipid profiles. blood pressure. Dark chocolate consumption (containing 180 mg polyphenols) resulted in reduction of total and LDL cholesterol among elevated serum cholesterol subjects [69]. 67. They suggested that specific insulinogenic amino acids may explain their findings. Brand-Miller et al. To a greater extend. 74]. dark chocolate administration ameliorated insulin [73. flavored milks and puddings) increased postprandial insulin secretion compared to strawberry flavour. medium. Low doses of dark chocolate (containing 30 mg polyphenols) supplementation to prehypertension subjects for 18 weeks significantly reduced systolic and diastolic blood pressure compared to polyphenols-free white chocolate [58]. cocoa powder supplementation in healthy males significantly prevented LDL oxidation [57]. total antioxidant capacity. Cocoa liquor showed dose-dependently prevents the development of hyperglycemia in diabetic obese mice [70]. In addition.Molecules 2008. and monomers. our previous studies indicated that 4 weeks of cocoa powder extract supplementation to diabetic animal model showed hypolipidemic and hypoglycaemic properties [71. However. and high dosages of cocoa powder supplementation. Mathur et al. cakes. subjects showed decreased plasma LDL cholesterol. oxidized LDL and apo-B concentration compared to baseline. The negative outcomes could be due to several factors which include the age of subjects. A growing number of studies were done on cocoa polyphenols and their protective effect towards LDL oxidation as an early indicator for the development of cardiovascular diseases [53. Cocoa powder supplementation significantly reduces the incidence of prostate carcinogenesis compared to positive . Cocoa powder exerted anti cancer properties in in vivo studies. body weight and body mass index. [67] indicated that after 4 weeks of low. There was also significant reduction in systolic blood pressure. soluble cellular adhesion molecules.

Daily cocoa extract administration prevented the overproduction of free radicals after heat exposure and thus protect from cognitive impairments [80]. Caffeine Theophylline Theobromine Dark chocolate is high in theobromine and caffeine due to the addition of high amount of cocoa solids than that of milk and white chocolates. Later. To the greater extend. These compounds. Methyxanthines present in cocoa [86]. It was initially found as a mixture of caffeine and catechin known as “caffeine-kakaool”. Bisson et al. Methylxanthines Apart from polyphenols. The supplementation also increased the life span of the tumor-bearing rats. [78] reported that cocoa powder dose-dependently decreased prostate hyperplasia through reducing dihydrotestosterone level and prostate size ratio. while the caffeine content is about 0. The structures of methylxanthines present in cocoa are shown in Figure 4. constituting about 4% on a fat free basis.Molecules 2008. namely caffeine. found in dark chocolates. Caffeine and 3-methylxanthine are the major alkaloids in mature pericarp. The amount of theobromine is higher. long-term supplementation of cocoa powder improve cognitive performance in aged rats compared to unsupplemented rats [79]. Figure 4. theobromine. Theobromine is the major methylxanthine present in cocoa. are responsible for chocolate cravings [81].2% [83]. and theophylline [11. In contrast. Caffeine was found in cocoa beans in 1909 [19]. Theobromine is also a major alkaloid in young pericarp and is present almost exclusively in the cotyledons of the beans. compared to . 12]. Pura Naik [84] has confirmed that theobromine is the dominant purine alkaloid present in cocoa beans. Forsyth indicated that caffeine can form a loose complex with epicatechin [82]. theophylline was present in low amounts [85]. Forsyth and Quesnel [24] indicated that only theobromine was identified in cocoa beans. 13 2197 controls [77]. cocoa is also rich in methylxanthines.

Kelly [87] has suggested that the contribution of theobromine in dark chocolate towards health benefits should be considered. 12. compared to those found in coffee and tea [15]. the possible synergistic interactions between flavonoids and methylxanthines are also unclear and need further study. it had been reported that adenosine receptor stimulates glycogenolysis in hepatocytes through binding to A2 receptor [97. theobromine. theobromine. caffeine supplementation had adverse effects on glucose metabolism and impaired postprandial glucose response [92]. Furthermore. It was noted that theobromine was responsible for the activation of . Graham et al. but are also rich in methylxanthines (caffeine. 90]. and theophylline). and C-peptide compared to decaffeinated coffee among healthy men [91]. but the question of whether the presence of methylxanthines enhances or reduces the health benefits of the cocoa flavonoids remains unanswered. the studies on the effect of caffeine on glucose metabolism have also demonstrated conflicting outcomes. The presence of methylxanthines may thus positively affect glucose metabolism [94]. 96]. methylxanthines particularly caffeine. Caffeine supplementation enhanced net hepatic glucose uptake through increment of glucose-6phosphate production in the liver during glucose load [14]. [101] indicated that aminophylline supplementation decreased glucose production with concomitant increased in insulin secretion. Theobromine is a psychoactive compound without diuretic effects. it should be noted that cocoa and cocoa products are not only rich in polyphenols. This is probably due to the dosage used as the theobromine content in the first study was almost three times higher than in the later one. Mechanistically. Administration of 5 mg caffeine/kg body weight reduced insulin-stimulated glucose uptake in T2DM and sedentary human subjects as measured by hyperinsulinemic-euglycemic clamp procedure [12. For instance. and caffeine. 98] and inhibit insulin secretion through binding to A1 receptor [99.Molecules 2008. methylxanthines inhibited phosphodiesterase and hence increase the intracellular concentration of cyclic adenosine monophosphate. [93] showed that the same dose of caffeine could also increase serum insulin and C peptide compared to placebo. 100]. To date. as the evidence in favor or against is often contradictory. Most of the studies underline the effects of polyphenols on the studied subjects. insulin. Previously. Caffeine levels are relatively low in cocoa. indicating aminophylline inhibits endogenous glucose production in type 2 diabetes. [89] have reported that theobromine and caffeine were neither prooxidant nor antioxidant. no effect on lipid profiles was observed after administration of cocoa extract. Although most of the studies indicated that the health benefits of cocoa or cocoa products were attributable to polyphenols [5]. but Vinson et al. Eteng et al. It was known that intracellular concentration of cyclic adenosine monophosphate is involved in the regulation of both insulin secretion from pancreatic cells and liver glucose output. and theophylline exerted antioxidant activity and protective ability under physiological conditions [88]. could exert prooxidant properties. 58]. compared to pure theobromine. Eteng and Etarh [102] demonstrated that single doses of theobromine (700 mg/kg body weight) significantly reduced lipid profiles in hyperlipidemic rats. [103] reported that supplementations of 3% and 15% cocoa powder that contained 56 to 265 mg theobromine to the rats significantly reduced body weight and also decreased lipid profiles. These results indicated that cocoa possessed hypocholesterolemic properties. In diabetics. Arias et al. Ingestion of 5 mg/kg body weight caffeinated coffee resulted in increased area under the curve for glucose. However. in contrast to the first study. 13 2198 caffeine in cocoa and cocoa products [11. Aminophylline (a type of caffeine metabolite) has been shown to stimulate glucose and arginine-stimulated insulin release [95.

Theobromine supplementation causes mortality in rabbits. respectively [119].5% of theobromine supplementation causes mortality in mature and immature rabbits and the effects were reported to be both dose. there are few studies on the contribution of cocoa peptides towards health. and failure in the release of late spermatids in male rats [13]. due to cardiac failure. theobromine intakes had been reported to have negative effects on reproductive health. A study has demonstrated the positive effects of theobromine on cancer. Of these. Theobromine was reported to potentially inhibit angiogenesis induced by ovarian cancer cells through inhibition of vascular endothelial growth factor production [105]. Cocoa is also rich in peptides and amino acids as described in the previous section.1 kDa [120]. while the globulins have molecular weights of 47. 118]. hydrophobicity of peptides also appears to be an important factor for their antioxidant activity. Cocoa beans contain four types of proteins. theobromine intakes among men have been associated with increased risk of prostate cancer [109]. Cocoa peptides are generally responsible for the flavour precursor formation [114-116]. Peptides Besides polyphenols and methylxanthines. Theobromine (250 mg theobromine/kg body weight) can lead to vacuolation within the Sertoli cells. In addition. albumin constitutes the major protein fraction [117. Soffietti [110] indicated that 1 and 1. [121] reported two new compounds were formed from vicilin protein during cocoa fermentation. and the albumin fraction has a molecular weight of 21.2 kDa. Buyukpamukcu et al. In contrast. caffeine from coffee or cocoa beverages is freely absorbed through the placenta and eventually leads to fetal growth retardation [112. namely albumins. Strachan and Bennett [111] did indicate sudden death of laboratory animals after theobromine administration. cocoa is also rich in proteins. In pregnant women. and its analogues pentoxyfylline. tyrosine. At a therapeutic dose of 500 mg/kg it has been used in the treatment of cardiac oedema and angina pectoralis. Previously. Albumin and globulin fractions accounted for 52% and 43% of total bean proteins. The storage protein comprises two vicilin fractions with molecular weights of 47. The supplementation caused changes in weight patterns and in the morphology of the thymus in both sexes of rabbits. due to . highdose cocoa extract containing theobromine could alter testis structure to a greater extent to that of pure theobromine. 113]. Bioactive peptides were reported to possess antihypertensives. Cocoa bean is the first to have vicilin-like globulin with sedimentation coefficient of 7-8S and a molecular weight of 150 kDa. and antiobesity effects [122]. Of these. Albumin has a molecular weight of 19 kDa. To the best of our knowledge. globulins. 13 2199 hormone-sensitive lipase. The peptides are nonapeptide and hexapeptides (a product of nonapeptide degradation). prolamin.and time-dependent. and glutelin. and 14.5 kDa. It has been found that peptides and amino acids are responsible for the taste and aroma precursors of chocolate. Marcuse [123] reported that the antioxidant activity was attributable to histidine. suramintheobromine and lisophylline are currently being exploited for cancer chemotherapy [106-108].Molecules 2008. hypocholesterolemic and hypotriglyceridemic. 31. Moreover. which hydrolyzed triacylglycerols and release free fatty acids and glycerols from adipose tissue into plasma [104]. antithrombotic. altered spermatid shape. histidine possessed strong radical scavenging activity due to the decomposition of its imidazole ring [124]. methionine and cysteine.1 and 39.

A prospective study (European Prospective Investigation Into Cancer and Nutrition (EPIC)–Potsdam Study) indicated that magnesium may reduce diabetes risk [132]. Magnesium was also found to be positively associated with insulin sensitivity in Insulin Resistance Atherosclerosis Study [129]. It is therefore possible that high magnesium content could partially contribute towards the health benefits of cocoa and cocoa-based products. cocoa powder provides 53% of DRI as per serving size (44 g) followed by dark chocolate (12%). milk chocolate and chocolate cake. The highest quantile (median intake = 457 mg/day) of magnesium intake in Health Professionals Follow-up Study was associated with reduced risk of developing cardiovascular disease compared to lowest quantile (median = 269 mg/day) [128]. milk chocolate (7%). Joo et al. fatty acids) [125]. Cocoa and cocoa products are also rich in copper and can contribute significantly to daily copper intake. Dark chocolate is the largest contributor of copper intakes in the US followed by chocolate milk. [133] showed that intracellular magnesium deficiency leads to the development of insulin resistance. 13 2200 increased interaction with hydrophobic targets (e. 74]. milk chocolate (24%). It was observed that magnesium intake from foods inversely related to blood pressure compared to intake from supplements [127]. [136] indicated that copper in cocoa and chocolate had significantly contributed to human diet. Moreover. Moreover. red wine. the health effects of peptides in humans and the optimal plasma levels remain to be elucidated [122]. In human subjects with hypertension. sweet chocolate (12%). 135]. dark chocolate administration ameliorated insulin [73. a positive association was found between total copper intakes and consumption of chocolate [136]. However. Kandeel et al. cocoa drink (1%). . Cocoa and cocoa products contained relatively higher amount of magnesium compared to black tea. Moreover.g. white chocolate (3%) and cocoa drinks (2%). chocolate syrup. Hence. and apples [15]. Previous studies seem to underestimate the contribution of minerals and peptides in cocoa and cocoa products towards health benefits. and white chocolate (1%). 131]. epidemiological study indicated that magnesium was inversely related to the development of metabolic syndrome [130. Based on Dietary Reference Intakes (DRI) of magnesium for men and women with an age range between 30-71 years old.Molecules 2008. and minerals. According to Dietary Reference Intakes. copper could be considered as one of the health contributors present in cocoa and cocoa products. Hence. Superoxide dismutase is a metalloenzyme (copper may be located in the center of the enzyme structure) that is involved in the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide [134. [126] showed that minerals are one of the important components in cocoa and cocoa products. cocoa powder (44 g) provides 189% of daily copper intake followed by dark chocolate (34%). Steinberg et al. The amount of magnesium (440 mg) in this study is almost equivalent to magnesium content in 88 g unsweetened dry cocoa powder [15]. peptides. Minerals As discussed. which approximately delivers 36% of DRI per day. The dose of dark chocolate (100 g) given contains 115 mg magnesium. sweet chocolate (28%). the health properties of cocoa and cocoa products are not solely dependent on their polyphenol contents. questions arise as to whether there is any direct contribution of magnesium besides polyphenols to health benefits? Magnesium is an important mineral in the regulation of blood pressure. but also contributed to by other components such as methylxanthines (caffeine and theobromine).

mousses and crème.Molecules 2008. followed by beans from Ghana and Trinidad [143]. The quantity of the flavonoids present in these products solely depends on the amount of non-fat cocoa solid content. white chocolate is prepared with cocoa butter and sugar without cocoa powder. and drinks. cocoa butter. and cocoa drink (1%). cocoa beans from different origins contain different amount of (–)-epicatechin and (+)catechin. thioredoxin reductase. Based on the significant contribution of selenium to antioxidant enzyme. fermentation. Cocoa products are products that were prepared from cocoa or cocoa-related components namely cocoa liquor. there are chocolates producers who produced chocolates from high-flavonoids beans from Ecuador and utilized special roasting methods that preserve flavonoids in the cocoa beans [141]. it works by detoxification of hydrogen peroxide and organic peroxides [135]. Most of the intervention studies used dark chocolate. Moreover. while white chocolate will have none. dark chocolate (3%). iodothyronine deiodinases. Countries of origin Cocoa from different varieties exhibited differences in polyphenols content by up to 4-fold [142]. They found that the highest phenolic content was in Malaysian beans followed by Sulawesian. it is important to consider the contribution of selenium in cocoa and cocoa products. This is due to the fact that dark chocolate contains more non-fat cocoa solid (cocoa powder) than other chocolates. Selenium exhibited antioxidant properties by fully or partially restoring antioxidant enzymes in rat tissue [139]. and reduced heat and alkalization treatments [141]. cocoa powder (44 g) could provide 11% selenium followed by milk and white chocolate (4%). Azizah et al. Cocoa beans from Ecuador possessed the highest amounts of (–)-epicatechin and (+)catechin. There was about 6-fold variation in epicatechin contents in fermented cocoa beans from different regions [40]. On the other hand. Moreover. Cocoa beans undergo various stages of processing before turning into raw cocoa (fermented and dried cocoa beans) and cocoa-based products. dark and milk chocolates are expected to have flavonoids. These chocolates are produced by controlling bean selection. and the products ranging from chocolates. the selection of the best quality cocoa and cocoa products could deliver the best antioxidant flavonoids. selenophosphate synthetase. Thus. 13 2201 According to the USDA database [15]. a high-flavonoids chocolate can be produced that preserves up to 70% of the flavonoids present in the finished product. Furthermore. Krawczyk [141] showed that flavonoid . A study reported that low level of selenium is directly related to diabetic nephropathy [138]. selenium exerted antioxidant ability through free radical scavenging and inhibition of lipid peroxidation [140]. [144] also reported that cocoa beans from different countries may have different polyphenols content. By controlling the process involved in preparing the chocolates. Factors affecting the quantity and quality of polyphenols in cocoa beans and cocoa-based products Recently. cakes and pastries. selenoprotein P and other selenoproteins [137]. Specifically. sweet chocolate (2%). Selenium is an essential micronutrient as cofactor in the formation of glutathione peroxidases. Thus. Ghanian and Côte d’Ivore. various types of dark chocolates are available in the market with high flavonoid contents.

External fermentation primarily involves the catabolism of the sugar pulp by microorganisms. about 50 and 60% of enzyme activity after first and second days is still remained. 142. According to Wollgast and Anklam [156] fermentation is an essential stage that lasts from five to six days in the development of suitable flavour precursors. In addition. catechin. high temperatures and prolonged processing . However. 158]. while internal fermentation encompasses the biochemical changes in the cotyledon of the beans [145. the level of flavonoids is also dependent on the processing steps with focus on the fermentation. The change has of great importance in determining the flavour of the final product [82]. Catechin has a bitter taste with a sweet aftertaste or is described as bitter and astringent [153]. and amount added in the production of the products. origins. Fermentation will reduce the level of bitterness and astringency of the cocoa bean which could be attributed to the loss of polyphenols during fermentation [40. Moreover. 13 2202 content of cocoa products depends on the cocoa beans used to make them. Stark et al. The production of aroma precursors during fermentation is important for producing the full aroma of chocolate [145. This step is crucial in determining the quality of cocoa aroma. Fermentation Fermentation is one of the steps involved in the production of cocoa beans. polyphenols diffuse from their storage cells and undergo oxidation to become condensed high molecular compounds mostly insoluble tannins. 149]. The conversion of simple cyanidin compounds to more complex leucocyanidin is the main change in the polyphenolic compounds in cocoa cotyledons. approximately 10 to 20% of epicatechin and other soluble polyphenols are reduced during fermentation. [143] reported that (–)epicatechin and (+)-catechin increased in the order of fully fermented (brown color). [154] reported that the bitter taste and astringency were not just attributable to polyphenols. Oxidation of polyphenols to insoluble tannins during fermentation was responsible for the formation of flavour precursors for chocolate processing [150-152]. for example type of cocoa bean. Research has shown that chocolates produced from unfermented beans have no chocolate flavor and are excessively astringent and bitter [148]. procyanidin B2. partly fermented (violet color) and unfermented (slaty color). This could also due to the diffusion of polyphenols into fermentation sweating [40. [154] showed that catechins. which include epicatechin.Molecules 2008. It is important to know the factors which influence the polyphenols content of finished products. were the major compounds responsible for bitterness and astringency of roasted cocoa. Furthermore. Caligiani et al. respectively [157]. This process involves both nonenzymatic and enzymatic. but were also contributed to by amino acids. There are internal and external fermentation stages involved during cocoa fermentation. numerous studies have been reported on the loss of compounds in cocoa beans during these processes. Fermentation is considered as one of largest influences on flavonoid levels in chocolates [141]. Pettipher [155] demonstrated that procyanidins are converted to largely insoluble red-brown material resulting in the characteristic colour of chocolate during fermentation and roasting. Stark et al. procyanidin B5 and procyanidin C1. To date. 147]. The aforementioned factors are directly or indirectly related to the quantity and quality of polyphenols content of cocoa and cocoa products. 146]. 157. and catalyzed by polyphenol oxidase. Even though this enzyme is strongly inactivated during the first days of fermentation. During fermentation.

dried. However. and roasted cocoa beans. the manufacturers tend to prefer Ghanian cocoa beans. Different degrees of roasting significantly increased the amount of (+)-catechin due to the isomerization of (–)-epicatechin. the ratio and types of polyphenols found in cocoa beans are unlikely to be the same as those found in the finished products [31]. and milk powder are used in making different types of chocolates namely dark chocolate. cocoa butter. or even within a region. Due to these factors. The introduction of heating during manufacturing of chocolates and cocoa-based products can change the enantiomeric composition of (+)-catechin [162]. sugar. other sweeteners. Polyphenols content gradually decreased upon fermentation from days 0 to 8 [153]. Therefore. Chocolates may have different percentages of non-fat cocoa solids. Furthermore several phenolic compounds were undetected in cocoa powder produced from fermented. 13 2203 times will decrease the amount of catechins [156]. and process of chocolates making. Tomas-Barberan et al. It has been shown that two days of sun-drying of fresh unfermented cocoa beans (without fermentation) causes a 50% decrease in epicatechin content. which are considered as less fermented and have low quality cocoa flavor [161]. oil. Alkalization (or dutching) of cocoa powder will reduce the polyphenol content and antioxidant activity [48. [4] quantified the content of dimers and trimers in unfermented and unroasted cocoa powder. milk crumb and emulsifiers. milk powder. During fermentation. epicatechin content was observed to decrease sharply. The bitterness of the chocolates was also due to the presence of flavonoids. The content of polyphenols can vary tremendously depending on the source of beans. cocoa manufacturers would blend the unfermented. Manufacturing process Chocolate is made from different recipes and contains other ingredients in addition to cocoa butter and powder [163]. sugar. most of the beans used for chocolates manufacturing are fermented. to obtain the desired flavour characteristics and also to reduce the excessive astringency and bitterness. In addition. yet. it is far from being a standardized process throughout the world. manufacturers tend to remove them in large quantities to enhance taste quality. Almost 90% of polyphenols are lost from fresh cocoa beans during fermentation. As cocoa powder is derived from fermented. dried. milk chocolate. [159] demonstrated that the high temperature used during drying of fermented cocoa beans had reduced polyphenol contents as a result of enzymatic oxidation. Non-enzymatic oxidation of polyphenols could also occur at this stage. . which could indicate that it is either being used up for the formation of large tannins or lost in the fluids that drain away [40]. and roasted beans compared to cocoa powder produced from unfermented beans [4]. primary and secondary processing conditions. between days two and three.Molecules 2008. the epimerization of catechins could be caused by the conditions applied during the extraction procedures. Dimers are present in cocoa powder produced from fermented and dried cocoa beans. This process may reduce the epicatechin content gradually during the process of making chocolates starting from fresh cocoa beans. There are different percentages of cocoa butter. and white chocolate. Kyi et al. which are well-fermented and flavorful than that of Dominican or Indonesian beans. Thus. the flavonoid content may not be detected in cocoa beans. partly fermented and fully fermented beans. 164]. In practice. the loss of phenolic compounds is higher than that of cocoa liquor [160]. The basic ingredients required for the manufacturing of chocolate are cocoa liquor. Apart from this.

semi sweet chocolate (15-19%). the higher amount of non-fat cocoa solid indicates the higher phenolic content in the chocolates. cocoa polyphenols contain different structures of flavonoids. and polymerization. Epicatechin has been reported as the main polyphenols present in cocoa beans [40. the presence of other nutrients (protein. and chocolate syrup (5-7%). typical RP-HPLC analysis was limited in separating enantiomers of catechin and epicatechin. milk chocolate (5-7%). thus creating difficulties in the determination of polyphenols content in chocolates. resulting in low plasma concentration of (–)-catechin. However. As reported. Furthermore. [161] showed that the highest NFCS was in cocoa powder (72-87%). According to Donovan et al. carbohydrate. followed by baking chocolate (45-49%). especially the chocolates that are high in total polyphenols. Monomeric flavonoids are absorbed in the small intestine. glycosylation. Furthermore. In addition. 13 2204 According to Cooper et al. [169] reported that (+)-catechin was 100 times more efficient than quercetin in an in vivo oxidative stress model. the presence of non-fat cocoa solid (NFCS) is an excellent marker to determine the total phenolic content. [168]. there was positive and significant relationship between NFCS and antioxidant properties [161]. This may overestimate the polyphenols content in certain type of chocolates. The presence of the (– )-catechin in chocolates could be due to epimerization of the (–)-epicatechin during the manufacturing of chocolates. sulfated. Besides physiological factors. Cooper et al. dark chocolate (20-30%). it does not correlate well with the cocoa and polyphenols content. acylation. The compound was formed during manufacturing process through epimerization of (–)-epicatechin and its epimer (–)-catechin [46]. Usually. or glucuronidated in the liver [167]. since it includes polyphenol-free cocoa butter. Miller et al. Bioavailability of cocoa polyphenols Recently. The relationships suggest that polyphenols are affected to the same degree. bioavailability of (–)-catechin was less than the (+) form of catechin. but polymeric procyanidins may be degraded by intestinal and colonic microflora followed either by absorption of the metabolites or excretion in the feces. food matrix and texture. The bioavailability of polyphenols greatly depends on the chemical structure. [5]. Different forms of catechins either (+) or (–) are absorbed differently. After absorption. there have been two major approaches commonly used to determine the availability of phenolic compounds either by measuring their concentration in plasma and urine after ingestion of known amount of foodstuffs or ingestion of the pure compounds [167]. Fraga et al. This may follow the agreement that epicatechin is well established as the main polyphenols found in cocoa beans. the monomers and dimers may be methylated. [89] reported that the amount of polyphenols increased in the order of hot chocolate < milk chocolate < dark chocolate < cocoa. Dark chocolates contain the highest NFCS among the different types of chocolates. A recent study showed that roasted cocoa beans and cocoa products contained flavan-3-ol (–)-catechin along with (+)-catechin and (–)-epicatechin. Vinson et al. conjugation. and fat) and the interaction between them may directly or . [165] reported that the percentage of cocoa which appears on chocolates' labeling cannot be used accurately to estimate the polyphenol contents. A study showed that fresh cocoa beans contain (+)-catechin with undetectable levels of the (–) form [162]. such as dark chocolates [165]. the concentration of (–)-catechin was higher to that of (+)-catechin in chocolates. On the other hand. 166]. Theoretically. Epicatechin has a strong correlation with other polyphenols compared to catechin [32].Molecules 2008.

the transferred dimers resulted in unmetabolized/unconjugated epicatechin monomer. The absorption of these glycosides is dependent on the position of sugar linkage [176]. Moreover. It was reported that plasma concentration of glucuronide conjugates of non-methylated and methylated (–)-epicatechin were higher compared to other forms. and sulfate-glucuronide (mixture of sulfate and glucuronide) conjugates.Molecules 2008. 13 2205 indirectly affect the bioavailability of polyphenols. (–)epicatechin and (+)-catechin can be detected as early as 0. detected that epicatechin increased markedly after chocolates consumption.5 h and reach maximal concentrations by 2 hr after acute consumption of cocoa [171]. In a human clinical trial. whereas aglycones (without sugar moiety) can passively diffuse through the small intestine. Baba et al. This indicates that epicatechin is absorbed from chocolates and is rapidly eliminated from plasma. However. (–)-epicatechin was detected as early as 0. Attainable plasma levels were 0. Excretion of (–)-epicatechin . As reported by Spencer et al. Baba et al.2 nmol/L after consumption of the procyanidins and then returned to normal levels [170]. most flavonoids are present as glycosides (attached to a sugar moiety).7 μmol/L (free epicatechin and epicatechin conjugates) from 80 g of black chocolates which contain 164 mg of epicatechin. [172] indicated that administration of procyanidins increased plasma (–)-epicatechin and (+)-catechin by 81% and 28%. respectively. The maximal concentration and area under the curve of plasma kinetics correlate well with the dose of chocolates. rather than methylated forms. There is a significant reduction in total (–)-epicatechin metabolites after 6 hr and the remaining conjugates are mostly present as the O-methylated form. [175] indicated that administration of procyanidins B2 [epicatechin-(4β-8)epicatechin] extracted from cocoa powder was absorbed in the plasma and excreted in the urine. conjugation. Using a pure (–)-epicatechin compound. acylation. or methylated forms [173]. Da Silva [178] showed that (–)-epicatechin is present in plasma in the forms of glucuronide and sulfateglucuronide conjugates (free and O-methylated) forms. Different types of polyphenols. This glucuronidation compound showed low antioxidant activity compared to intact compounds as it has lost the catechol structure of the B ring responsible for the antioxidative effects. [173] reported that (–)-epicatechin from chocolate or cocoa are present in plasma of human volunteers as sulfate. Murphy et al. the administration of 148 mg of procyanidins had increased plasma epicatechin at 2 h compared to baseline (0 h) [170]. To a greater extend. glucuronide. respectively. The glucuronidation of (–)-epicatechin occurs at the position 3‘ of the B ring in humans [179]. A study related to plasma kinetics of epicatechin in men after consumption of 40 g and 80 g of dark chocolates. glucuronides. The compounds appeared maximally in the plasma at 30 min and decreased gradually from 30 min to 300 min. reaching a maximum between 2-3 hours [174]. and glycosides all showed different degrees of absorption. In addition.5 to 1 hr after chocolate or cocoa consumption and they are present mainly as sulfate conjugated. plasma procyanidin dimer. [179] found that (–)-epicatechin metabolites present in human plasma are different than the forms present in rats. Glycosidic flavonoids require hydrolysis by colonic microflora to cleave the sugar and release the aglycone for absorption. With the exception of catechin and epicatechin. glycosylation. polyphenol structures (+ or –). plasma epicatechin concentration increased to 21. Similarly. Natsume et al. [177] only small amounts of procyanidins B2 and B5 were transferred to the serosal side of enterocytes in isolated small intestine. The glucuronidation of (–)-epicatechin occurs at the 7 position of the A ring and the 3’ position of the B ring (Figure 1) in rats and humans. (–)-Epicatechin metabolites are present in urine within 24 hr in the range of 25% to 30%.

5-(m-hydroxyphenyl)valeric acid. it is clear that under the acidic environment in gastric milieu. The presence of these monomer and dimer could in turn enhance their absorption in the small intestine [190]. To date. procyanidins oligomers are hydrolyzed to monomeric epicatechin and dimer. 2-(m-hydroxyphenyl)acetic acid. and their dimers was influenced by physiological pH. and thus available for absorption or metabolism.3 μM) compared to tumor-bearing (10. protein is one of the food factors that was widely studied due to their interaction with polyphenols. Moreover. the evidence for the interference of protein with polyphenols remains questionable. and phenylpropionic acid after 48 h incubation with human colonic microflora. Zhu et al. Moreover. [187] reported that the degradation of catechin. the nature . vanillic acid. 2-(p-hydroxyphenyl) propionic acid.6 μM) rats [191]. Hence. very little study has been done to investigate the effects of food components in our diet on the catabolism products of unabsorbed polyphenols in the guts because most of the high molecular weight polyphenols are not absorbed in the small intestine. there is a difference in the metabolism of polyphenols between normal and disease-state subjects. It has been demonstrated that polyphenols bind to salivary protein and resulted in precipitation of insoluble complex and astringent flavour [184]. ferrulic acid. Serafini et al. epicatechin. and m-hydroxybenzoic acid [33]. In addition. For instance. The presence of protein in food matrix may form complexes with polyphenols [181]. 2-(m-hydroxyphenyl)propionic acid. [17] however found that bioavailability of polyphenol monomer (epicatechin) was not reduced when cocoa is ingested with milk. Protein mainly from milk (chocolates) and digestive environment (salivary protein) may form complexes with polyphenols and reduce their bioavailability [18]. Relatively little studies have been reported on the bioavailability of flavonoids in subjects with disease. Polyphenols not only bind to salivary protein but also with dietary protein and digestive enzymes in which in turn may influence their transportation and absorption activities [185]. most of the results were based on the normal and healthy subjects where the rate or extend of absorption are assumed to be normal. The presence of protein either in foods matrix or in the digestive system could at least affect polyphenols bioavailability.Molecules 2008. [182] demonstrated that the presence of milk inhibits polyphenol absorption. mhydroxyphenylacetic acid. Rios et al. For example. [186] and Klimczak et al. [189] showed that polymeric proanthocyanidins were degraded into low molecular weight aromatic compounds. namely m-hydroxyphenylpropionic acid. antioxidant capacity of polyphenols was modified with the presence of protein [183]. namely. it was observed that the level of (–)-epicatechin metabolites excreted in urine was in the close range after equivalent ingestion of pure (–)-epicatechin and (–)-epicatechin from cocoa powder. 3. [188] indicated that autooxidation and epimerization were two major reactions involved in determining the stability of phenolic compounds under typical experimental conditions. 2-(p-hydroxyphenyl)acetic acid. However. However. Deprez et al. Schroeter et al. high molecular weight polyphenols may form strong interaction with protein. the bioavailability of (–)epicatechin was not influenced with the presence of other compounds present in cocoa powder. Studies by Zhu et al. 13 2206 metabolites in urine was observed to be dose-dependent [180]. Hence. The concentration of naringenin metabolites was higher in healthy (17. In addition. flavanol-rich chocolate intake increased urinary excretion of phenolic acids.4-dihydroxyphenylacetic acid. [33] reported that procyanidins were remarkedly stable in the stomach environment. a study indicated that the addition of milk reduces the antioxidant capacity by 30% [18]. Similarly. However. Moreover.

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